Wnt signaling pathways regulate cell proliferation, differentiation, polarity, and behavior in embryonic development, in adult stem cells, and in cancers and other disease states. Wnts are therefore reasonable candidates for playing essential roles in the normal biology of human embryonic stem cells (hESCs) and for altering the proliferation and differentiation of hESCs cultured in vitro. At least two Wnt signaling pathways exist in vertebrates, the Wnt/li-catenin pathway that controls gene expression through a well-defined pathway, and one or more "non-canonical" pathways that can antagonize the Wnt/B-catenin pathway. In various stem cells the Wnt/li-catenin pathway has been implicated in controlling self-renewal, proliferation, and differentiation. In contrast, the existence or functions of the non-canonical pathways in hESCs have not been reported. The overall goal of this project is to test the hypothesis that distinct Wnt signaling pathways are functionally involved in the in vitro proliferation and differentiation of hESCs, to elucidate the different mechanisms of signaling, and to leverage these insights to direct hESC differentiation in vitro. The specific aimsare: Aim 1. Interrogating distinct Wnt signaling pathways in hESC Aim 2. Regulation of Wnt pathways in hESCs by miRNAs Aim 3. Responses of hESCs to gradients of Wnt-3a and Wnt-5a ^ Collectively, the proposed studies will lend further insight into the mechanisms underlying the regulation of hESCs by Wnt signaling, particularly with regards to the role of Wnts in hESC proliferation, differentiation and motility. These findings can be exploited to manipulate hESCs in vitro with the eventual goal of generating protocols for hESC differentiation that can have therapeutic impact on human disease.